overview of telomeres & telomerase biology: clinical implications in cancer and aging meir lahav...

Overview of telomeres & telomerase biology: Clinical implications in cancer and aging

Meir Lahav MDLaboratory for telomere research, Rabin Medical Center, Beillinson CampusFelsenstein Medical Research Center 8 March 2010

Historical perspective

• 1908, McClintock & Muller“Chromosome bore a special

component at their ends that provided stability”

• Telomere: telos- end, meros- part

• 1961, Hayflick & Moorehead

“Normal somatic cells have a limited life span- a status that is terminated in M1 stage- replicative senescence”.

Leonard Hayflick

Biological landmarks

• 1971, Olovnikov: “Marginotomy”- the end-replication problem may account for the Hayflick limit

• 1972, Watson: DNA polymerase could not replicate chromosomes to the tip

The end-replication problem

5’ 3’3’ 5’

DNA Replication

5’ 3’ R R R R

3’ 5’R

RNA primer removal

Fill-in DNA replication

Ligation 5’ 3’

3’ 5’

Each division 50-100 bp loss

Biological landmarks (cont.)

• 1978, Blackburndiscovered telomeres in Tetrahymena (TTGGGG)n

• 1984, Blackburn & Greidertelomerase activity was detected in Tetrahymena

Telomeric end of DNA

Genomic DNA Telomere

Molecular structure of the telomere

Functions of telomere [(TTAGGG)n]

• Protects the chromosomal ends from: – Recombination– End-to-end fusion– Recognition as damaged DNA

• Enables a complete replication of the DNA • Contributes to the functional organization

of chromosomes in the nucleus• Participates in regulation of gene

expression • Serves as “mitotic clock”: shortens with

each cell division

Telomere length in healthy population Uziel et al. 2002

y = -27.45x + 6972.5

R2 = 0.4636

0

1000

2000

3000

4000

5000

6000

7000

8000

0 20 40 60 80 100

Age (years)

Tel

om

ere

len

gth

(b

p)

Consequences of telomere shortening & damage

Two-step hypothesis of cellular senescence and immortalization

Wright & Shay Microbiol Mol Biol Rev 2002

Telomerase

telomerase

5’ TTAGGGTTAG CAAUCCCAAUC

telomerase

5’ TTAGGGTTAGGGTTAG CAAUCCCAAUC

telomerase

5’ TTAGGGTTAGGGTTAGGGTTAG CAAUCCCAAUC

telomerase

5’ TTAGGGTTAGGGTTAG CAAUCCCAAUC

Telomerase

hTERT

hTR-CAAUCCCAAUC

Elongation of a telomere by telomerase.

Wong L S et al. Cardiovasc Res 2009;81:244-252

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Keeping telomerase in its place Maser & DePinho Nature Medicine 2002

The telomere model for cellular transformation

Germ cells: telomerase ON

Somatic cells:

telomerase OFF

Immortal cells:

telomerase ONOncogeneticallytransformed cells:bypass senescence,

telomerase OFFSenescence Crisis

# of cell divisions

Telo

mere

len

gth

TRF measurementsShapiro, Uziel and Lahav

2000

Southern blot FISH flow

FISH on paraffin embedded tissues

Clinical applications of telomere research

Senescence

Cancer

Acquired capabilities of cancer)Hanahan and Weinberg, Cell 100: 57-70, 2000(

Minimal set of genetic alterations required for conversion of fibroblasts to cancer cells Sun et al 2006

• Malignant conversion: – SV40 large T antigen (p53

and pRb inactivation) – Ras activation

• Malignant cells are not immortal - enter crisis and die

• Telomerase expression renders cell immortal

Telomerase up-regulation cause or consequence

• Human cancer cells have– shorter telomeres then normal– dysfunctional telomeres (anaphase bridges,

ends fusions etc.,)

• Correlation between anaphase bridges and telomere length

• Human colorectal cancers show a peak in anaphase bridges index in early lesions;

Effect of telomerase inhibition on malignant cells growth

Telomerase inhibition in cancer Lahav

2010

Chemosensitization by telomeres Lahav 2009

Comet assay DNA damage Lahav 2010

DNA damage focci telomere dysfunction Lahav 2009

Association of telomerase activity with disease free survival in non-small cell lung cancerGonzalez-Quevedo, R. et al. J Clin Oncol. 2002;20:254-262

actin

hTERT

IGFI-R

CD63

actin

[ThD] g/ml 0 12.5 25 50 100

hTERT

IGFI-R

actin

CD63

[ThD] g/ml 0 12.5 25 50 100

hTERT

IGFI-R

CD63

[ThD] g/ml 0 12.5 25 50 100 RPMI 8226 U266

ARH-77

Thalidomide downregulates telomerase promoter gene expression molecular pharmacologyDruker, Uziel, Lahav et al. 2004 molec pharmacol

0M 10M 15M R8

Inhibition range: 70-90% 0

20406080

100120

1 2 3 4 5

time (days)

telo

mer

ase

acti

vity

(% o

f co

ntro

l)

Kinetics of telomerase activity during Gleevec treatment

Telomerase activity after Gleevec 5 days treatment

0

20

40

60

80

100

120

1 2 3

[Gleevec](uM)

Te

lom

era

se

ac

tiv

ity

(%

)

0 10 15

Gleevec inhibits telomerase activity in SK-N-MC cellsUziel and Lahav,2005 BJC

Control cells STI571 treated cells

Telomerase cellular localization in STI571 treated cells

Uziel, Beery et al 2003

Telomerase as a drug target

• Significant difference of telomerase expression between malignant and normal tissues

• Possible adverse effects: damage to stem and germ cells

• Telomerase inhibitors will be effective only when the telomeres shorten to critical length

• Will probably be used as an adjuvant therapy

Potential effects of telomerase inhibition over time on telomere length and proliferative capacityExperts reviews in molecular medicine 2002

Strategies for inhibition of telomerase activity• Telomerase targeting

agents:– The RNA template– Reverse transcriptase

inhibitors– Modulators of telomerase

regulating proteins

• Telomeres targeting agents– Inhibitors that interact with

G4-DNA structures– Inhibitors against

telomeres associated proteins

– “Old” DNA -interacting drugs

• compounds from random screening

Effect of telomerase antisense on malignant cell cultureUziel and Lahav, 2004

Antimetastatic effects of GRN163L on pretreated A549-Luc cellsDikmen, Z. G. et al. Cancer Res 2005;65:7866-7873

0

20

40

60

80

100

120

0 5 10 15

Vincristine (ng/ml)

pro

life

rati

on

(%

)

0

20

40

60

80

100

120

0 50 100 150

Doxorubicin (ng/ml)

pro

life

rati

on

(%

)

Control

+GRN163

Telomere attrition sensitize SK-N-MC cells to DNA SS breaks inducing agent, CisplatinumUziel and Lahav, 2006

Telomerase inhibition – future directions

•New effective inhibitors

•Antitelomerase vaccines

•Antitelomerase adoptive immunotherapy

•Promoter driven therapy

•Development of antitelomerase – cytotoxic drugs – other biologic interventions combinations

Telomerase promoter-driven gene therapy

•hTERT promoter is highly active in cancer cells (not active in somatic cells)

•Expression of harmful genes under the control of hTERT promoter- expression directed to malignant cells

•Genes used– Proapoptotic genes: caspase 8, caspase 6, TRAIL,

Bax– Prodrugs– Viral lytic genes: adenoviruses

Adenovirus and telomerase promoter

Telomerase immunotherapy

• Immunizing patients against tumor antigens to elicit antibody or cytotoxic T-cells killing of tumor cells

• T cells against a short hTERT peptide in vitro and in mouse models in vivo; Somatic cells are not affected

• Prostate or breast cancer patients were vaccinated with cells expressing tert peptide; 4 responded; No se.

• 12 prostate cancer patients were treated as above, majority responded positively

Aging

Aging

Comparison between a single homologue from one individual and a single homologue from an unrelated individual carrying the same genetic marker

Dolly or failure of resetting the cellular clock

Willmut et al, 1997

Telomere length & survival rate

Trans-differentiation of pluripotent stem cells

Telomerase effect on cells

Telomere binding defect in progeria

Diabetes control and telomeres Lahav

2006

Copyright ©2010 American Heart Association

De Angelis, A. et al. Circulation 2010;121:276-292

Telomere-telomerase and p53 function

Histogram showing haemoglobin levels and their association with telomere length.

Wong L S et al. Eur J Heart Fail 2010;12:348-353

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2010. For permissions please email: journals.permissions@oxfordjournals.org.

Telomere length in the anaemic vs. non-anaemic group.

Wong L S et al. Eur J Heart Fail 2010;12:348-353

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2010. For permissions please email: journals.permissions@oxfordjournals.org.

Copyright restrictions may apply.

Farzaneh-Far, R. et al. JAMA 2010;303:250-257.

Absolute and Relative Mean Changes in Telomere Length Over 5 Years by Quartile of Omega-3 Fatty Acid Level, Adjusted for Age and Baseline Telomere Length

Translational applications ;

•Cancer; Mechanism of malignancy

• Therapeutic approaches

•Aging; Cellular ( stem cells)

• Organism ; normal

• accelerated aging